On Jun 25, 11:01 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <f5574569-d585-4f87-9c78-e2de610b5... from d1g2000hsg.googlegroups.com>, peter <peter.ianak... from gmail.com> wrote:
>>>> >On Jun 24, 2:02 am, d... from no.email.thankstospam.net (DK) wrote:
> >> In article
> > <8bd38140-d8fa-49b5-a790-af29f4484... from m73g2000hsh.googlegroups.com>, peter
> > <peter.ianak... from gmail.com> wrote:
>> >> >On Jun 23, 9:58 pm, d... from no.email.thankstospam.net (DK) wrote:
> >> >> In article <mailman.543.1214259080.3533.meth... from net.bio.net>,
> >> > hroyc... from nmsu.edu wrote:
> >> >> >Good question! I think long time ago ... in the last century ...
> > Maniatis
> >> >> >et al.'s Lab Manual described the specificity of these enzymes. I have
> >> >> >not really used dNTP for phosphoryltion, so I don't know how efficient it
> >> >> >would be. Structurally the two are similar enough.
>> >> >> I've never heard of any kinase being able to use dATP with any
> >> >> reasonable efficiency. I'd think that evolution worked out
> >> >> a way for things being pretty good at distinguishing oxy vs
> >> >> and desoxy ribose. Much like DNAse vs RNAse.
>> >> >> DK
>> >> >I thought so too, but it seems that DNA end repair by T4DNA pol +
> >> >T4PNK works fine just with dNTP (not supplemented with rATP). Strange
> >> >Peter
>> >> How exactly do you define "works fine"? And what kind of DNA
> >> is being end repaired? If it's genomic sheared DNA I would have
> >> thought just T4 poly would suffice, no need for PNK.
>> >Lambda sonicated DNA jugged by shift after self ligation.
>> That does not necessarily surprise me. Lots of break will be with
> 5' end phosphate anyway and others will have phosphates after
> the poly chews on them.
>> >Also PCR
> >product produced by Taq pol and blunted, then self ligated.
>> Yeah, this is surprising. Just a thought: naturally, ATP is
> present during ligation. PNK is very efficient enzyme and is
> always added in huge excess. If you don't heat kill after
> DNA repair and before ligation, then of course it is that
> you see a dependence on ATP during pre-ligation step. But
> even if you heat kill but some of it survives, there might be
> enough PNK surviving to phosphorylate PCR product ends.
>> An essential control would be thorough heat kill followed
> by complete deproteinization before the ligation. I still doubt
> PNK can use dATP.
>> DK
Well said. I also think dATP is not a PNK substrate, but the ultimate
proof will be to use gamma labeled dATP in kinase reaction. We shall
know soon.
-Peter
P.S. I am not heat inactivating PNK - just doing Qiagene purification
with 2x wash with binding buffer (6M Guanidine salt)